*2.3. Genomic Library Preparation and Sequencing*

Preparation of paired-end (PE) sequencing libraries of K133WT and K133AS-R was initiated with 200 ng of genomic DNA using a Truseq Nano DNA Library preparation kit (Illumina, Inc., SanDiego, CA, USA). The generated library was examined in a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using a high-sensitivity (HS) DNA chip and sequenced using the Illumina Hiseq 2000 platform according to the manufacturer's standard cluster generation and sequencing protocol [42]. Briefly, the mechanical shearing of gDNA by a Covaris instrument (Woburn, MA, USA) was done to generate fragments of 250–350 bp, after which fragmented ends were repaired and tailed with A at 3′ . Thereafter, adapters were ligated, which was necessary for binding dual-barcoded libraries to the flow cell for sequencing. Finally, 314–355-bp libraries were generated and high-fidelity PCR amplification was done using HiFi PCR master reaction component mix to ensure maximum yield from limited amounts of starting material for sequencing on an Illumina Hiseq 2000 platform (Illumina Inc., San Diego, CA, USA) (2 × 150 bp chemistry). Whole-genome sequencing resulted in the generation of approximately 4 GB data per sample. The sequences of *L. donovani* K133WT and K133AS-R are available with NCBI GenBank as a BioProject with SRA accession no. PRJNA657979.
